1. Field of the Invention
The present invention relates to columns and methods of making columns for separation techniques and apparatus. More specifically, the present invention provides a separation bed and method of making the same for use in various electromigration and non-electromigration separation columns, such as high-performance liquid chromatography, gas chromatography, capillary electrophoresis, capillary electrochromatography, and supercritical fluid chromatography.
2. Description of Related Art
Capillary electrochromatography or CEC is a fairly novel electrokinetic separation technique representing a hybrid of high-performance liquid chromatography or HPLC and capillary electrophoresis, known as CE. In CEC, the electroosmotic flow, or EOF is used to drive the mobile phase through the capillary, using typical HPLC mobile and stationary phases that provide the essential chromatographic interactions. Because of the flat plug-like profile of the electroosmotic flow, CEC offers greatly enhanced separation efficiencies relative to HPLC. Unlike CE, CEC is not restricted to charged solutes. Thus, the potential for CEC, as a separation technique, is much wider.
Capillary electrochromatography is a rapidly growing area in analytical separations. A great deal of research effort is currently being devoted to materialize the great analytical potential that this new hybrid technique has to offer. In order for CEC to achieve success as an independent chromatographic separation technique significant advancements are needed in the area of column technology. This is explained by the fact that in CEC, the column not only serves as the separation chamber, but also as the pumping device to drive the mobile phase through the system. This makes the column the “heart” of the CEC system both in the functional and literal sense of the word.
Two major types of columns are used in current CEC practice. These are packed and open tubular types. Packed columns comprise the predominant class of CEC columns. Most often the packed capillaries contain 1.5-5 μm, non-polar, octadecylated or ODS particles. The ODS particles possess both the chemically bonded octadecyl stationary phase, providing the essential chromatographic interactions, and the silanol moieties, responsible for the generation of electroosmotic flow to drive the mobile phase and the solutes through the packed capillary. The commercial availability of the ODS-bonded particles and the previously established liquid chromatography or LC separation protocols are two advantages attracting many researchers to use these packed capillaries in CEC. However, the most significant advantage of packed columns in CEC is the possibility of using small micrometer and nanometer size particles. High separation efficiency during fast analysis is achieved in packed CEC columns without requiring ultra-high pressures, as in HPLC to drive the mobile phase through the columns packed with the small particles.
The greatest challenge is the preparation of a uniform packing bed using the small particles. Researchers currently use a variety of packing procedures ranging from slurry packing, electrokinetic, centripetal, and supercritical fluid packing methods. These all involve a plurality of steps to effect the packing process and even with close monitoring do not give as uniform a bed as desired for many applications.
Furthermore, a great degree of difficulty still remains associated with the ability to pack long, narrow bore capillaries. In addition, most packed capillaries require end flits of a different material to retain the packing particles within the packed capillary bed. Creation of those flits remains to be a problem in column preparation as these flits must be rigid enough to retain the packing particles under a wide range of column packing, rinsing and operating conditions. Yet these flits must also possess a highly porous structure to permit a uniform mobile phase flow through the entire cross-section of the column. A further problem arises in that the presence of the frit material makes the packing in the column non-homogeneous due to the presence of a different material and this can cause problems with the separation characteristics of the final column.
Monolithic column technology can effectively overcome both of the difficulties associated with conventional packed capillary column technology. In the monolithic approach, a continuous separation bed is created inside the capillary tube using a solution, which undergoes both chemical and physical changes in the capillary environment to produce the separation bed. In addition, the choice of appropriate chemistry allows the porous bed to chemically bond to the inner walls of the capillary by a condensation reaction and the resulting packed tube is also homogeneous in nature.
The use of monolithic columns has been reported in gas and liquid chromatography and is also currently being used in CEC to alleviate the extensive labor involved with packed column fabrication. Moreover, the greatest inherent advantage of the monolithic capillary columns is the elimination of the need for the end frits. The elimination of these end frits allows the entire column to remain homogeneous, rather than exhibiting different properties by the packing particles and the end frits. It has also been demonstrated that the end frits reduce the column's separation efficiency and are responsible for bubble formation during the analysis.
Although much simpler than particle packed capillaries, monolithic columns derived by organic polymerization also possess certain limitations. One critical drawback associated with this type of monolithic capillary is the tendency of the polymer network to swell during exposure to certain organic solvents, which are contained in the running mobile phase. This swelling may result in reductions in the permeability of the monolith as a result of alterations in the porosity of the monolith. Such structural change ultimately leads to changes in the column performance during the course of its use.
Unlike the monolithic separation beds from organic polymers, columns-containing a porous silica-based monolithic matrix prepared through sol-gel chemistry do not suffer from the swelling phenomena thus offering a versatile and promising alternative to organic packed capillaries. In addition, monolithic columns, since they are prepared without end frits can produce a homogeneous separation column, which is highly desirable for a wide variety of separation techniques.
Pretorius was one of the first influential pioneers of CEC who, in 1974, demonstrated the advantages of electroosmosis as a pumping mechanism for chromatographic separations. Jorgenson and Lukacs published CEC analyses of 9-methylanthracene and perylene on an ODS-packed capillary column. Meanwhile, a 1987 report by Tsuda demonstrated the possibility of achieving CEC separations by the simultaneous use of both electroosmotic and pressure-driven flows in the separation column. Yet Knox and Grant made another significant contribution to the development of this technique. Following this publication, the term “electrochromatography” became generally accepted and numerous researchers refocused their attention to CEC.
As described earlier, two types of monolithic columns have been developed: (1) organic polymer-based and (2) bonded silica-based. In the first approach, fabrication of a monolithic capillary column is accomplished by polymerization reaction of organic monomeric precursor(s). Hileman et al used Carbowax coated open pore polyurethane monolithic capillaries for the separations of several classes of analytes including aromatic hydrocarbons, aliphatic alcohols and metal chelates through gas chromatography. Hjerten et al prepared monolithic capillaries with compressed polyacrylamide gels for separation of proteins using HPLC and of low molecular mass compounds and basic proteins using CEC. Frechet and coworkers reported a series of publications on the use of methacrylate monomers for the preparation of HPLC and CEC monolithic capillaries through copolymerization. Palm and Novotny prepared CEC monoliths using mixtures of polyacrylamide/polyethylene glycol, derived with either C4 or C12 ligands, which were used to separate alkyl phenones and peptides. Additionally, Fujimoto et al reported the usage of cross-linked polyacrylamides for the separation of small dansylated amino acids and neutral steroids on monolithic CEC capillaries.
An alternative to a column with an organic polymer-based stationary phase column is one with a bonded silica stationary phase prepared by sol-gel chemistry. Cortes and coworkers prepared porous beds by polymerizing potassium silicate solutions in situ. The columns containing the porous beds were then packed with 5 μm Spherisorb ODS particles for use in LC. Fields used solutions of potassium silicate and formamide to create a porous bed that was further reacted with dimethyloctadecylchlorosilane, and achieved plate heights of 65 μm in LC.
Tanaka and coworkers used the sol-gel technique for the development of an octadecylsilylated, porous monolithic column for use in LC. In this study, poly(ethylene oxide), PEO, was incorporated into a mixture of tetramethoxysilane (TMOS) and acetic acid to develop porous silica rods, followed by an in-column octadecylsilylation reaction. Following washings, and drying at 50° C. for three days, the silica rods were then treated for two hours at 600° C.
Dulay et al used sol-gel technology for the preparation of monolithic columns loaded with 3 μm ODS particles. The sol-gel solution served as a retaining matrix immobilizing and shielding the ODS stationary phase particles. Sol-gel capillary columns containing the ODS embedded particles yielded CEC separation efficiencies on the order of 80,000 plates/m (16,000 plates/column) for a test mixture of six uncharged polyaromatic hydrocarbons or PAHs.
Lee and coworkers also used sol-gel chemistry to glue 7 μm ODS particles thereby creating a continuous large-pore CEC column. The sol-gel technology in this approach was used to create a bridge between adjacent particles; as well as the capillary wall and particles in its vicinity, thereby eliminating the need for retaining end-frits, thus result being efficient separations of small organic and aromatic amine compounds on such “sol-gel-glued” monolithic columns.